Saccharomyces cerevisiae is an attractive organism for heterologous protein production and many suitable expression systems have been developed to produce high level of proteins with this organism. This yeast is also the best studied eukaryotic organism and many molecular tools have been developed. However, S. cerevisiae has some limitations in its commercial application, due to its relatively poor secretion efficiency of proteins, the need to use fed-batch fermentation techniques to attain high cell densities and hence improve the protein synthesis, and secretion of O- and N-glycosylated proteins which are often hyperglycosylated. Although, S.cerevisiae is regarded as a very proteolysis weak host organism, a further problem encountered in production of some heterologous proteins in S. cerevisiae is low yield, presumably due to proteolytic processing both in intracellular compartments and at the plasma membrane cf. Gabrielsen et al. Gene, 90:255-262, 1990, (secretion of human parathyroid hormone by S. cerevisiae), Rokkones et al., J. Biotechnol. 33:293-306 (secretion of human parathyroid hormone by S. cerevisiae), and Bitter et al. Proc. Natl. Acad. Sci. USA, 81:5330-5334, 1984 (secretion of .beta.-endorphine by S. cerevisiae).
According to Waldron and Lacroute, J. Bacteriol., 122:855-865,1975, the protein synthesis rate in yeast is dependent on the specific growth rate. They demonstrated that the net rate of protein synthesis in yeast lowered with decreasing specific growth rate. This was due to the decrease in average ribosomal efficiency i.e. the rate of protein synthesis per ribosome.
In batch culture with glucose as the carbon and energy source, the yeast S. cerevisiae will mainly ferment glucose to ethanol. Under anaerobic conditions, this is the only mode of energy production. In the presence of oxygen respiration occurs. However, alcoholic fermentation may s et in even under aerobic conditions if the glucose concentration surpasses a critical threshold value (Verduyn et al, J. Microbiol. Methods, 2:15-25, 1984; and vanDijken and Scheffers, FEMS Microbiol. Review, 32:199-244, 1986). This affects the biomass yield drastically (Reiger et al, J. Gen. Microbiol., 129:653-661, 1983; and von Meyenburg, Arch. Microbiol., 66:289-303, 1969). At the level of pyruvate, respiration competes with alcoholic fermentation via the mitochondrial pyruvate dehydrogenase complex and the cytosolic pyruvate decarboxylase. Acetaldehyde formed by the activity of pyruvate decarboxylase can after oxidation to acetic acid enter the tricarboxylic acid (TCA) cycle via acetyl CoA. Alternatively, acetaldehyde may be reduced to ethanol instead of being oxidized to carbon dioxide. S. cerevisiae can secrete all its fermentive metabolites acetate, pyruvate, ethanol, glycerol and succinate in glucose limited aerobic batch fermentation.
The overflow metabolism and repression of respiration in yeast strains resulting in redirection of substrate towards fermentative metabolism resulting in formation of ethanol and other primary products of fermentative activity, is referred to as the "Crabtree effect". This effect is remarkable in the species Saccharomyces cerevisiae. However, so far it has not been clear what the situation is with other species belonging to the genus Saccharomyces. Presently the genus Saccharomyces consists of more than ten species (Piskur, J. et al Int. J. System. Bacteriol. 48:1015-1024, 1998).
In literature, most of the yeast species employed for heterologous protein expression like Kluveromyces lactis, Pichia pastoris, Hansenuela polymorpha, Schwanniomyces occidentalis and Yarrowia lipolytica, are not respiration-limited yeasts (Kreger-van Rij, Classification of yeasts, in Yeast vol 1:5-66, 1987 and Heslot et. al., J. Bacteriol., 104:473-491, 1970). These strains differ from S. cerevisiae in being Crabtree negative yeast strains, thus having the advantage of utilizing the substrate more efficiently for protein and biomass synthesis. However, from a molecular biology aspect these yeast strains are not very well characterized and they are not as easy to manipulate as S. cerevisiae. Moreover, the secretion properties of these yeast strains are not characterized as well as for S. cerevisiae.
The present invention is based on the surprising recognition by the inventors hereof that at least one Saccharomyces species, namely Saccharomyces kluyveri is Crabtree negative or is only effected in a very small degree by glucose surplus during aerobic batch fermentation. S. kluyveri is a distant relative of S. cerevisiae and showed higher biomass yield on glucose than S. cerevisiae in batch fermentation confirming its higher respiratory capacity over S. cerevisiae. This is a desirable feature for the protein biosynthesis. FIG. 1a, 1b, 2a and 2b clearly demonstrate that the Saccharomyces kluyveri strains are "Crabtree negative".